JP5381119B2 - Method for producing saccharides from bark raw material - Google Patents

Description

  The present invention relates to a novel saccharification method using bark of a woody plant as a raw material.

  As one of the carbon dioxide emission reduction measures for suppressing global warming, bioenergy development obtained by converting biomass into energy is being carried out. As a method of biomass conversion, as shown in many books (Non-Patent Documents 1 to 4), pyrolysis, gasification, anaerobic fermentation, etc. are widely performed. Methods for obtaining ethanol by fermenting saccharides are widely studied. Ethanol can be used as a liquid fuel, particularly as a transportation fuel. In the United States and Brazil, a process for producing bioethanol using starch or sugar obtained from corn or sugar cane as a raw material has already been put into practical use. It has been pointed out that when the price of gasoline rises with these raw materials, corn, sugar cane, etc. that should have been originally produced for food are used as bioethanol raw materials. Therefore, bioethanol production using raw biomass that does not compete with food is required.

Unused biomass includes herbaceous biomass such as rice straw and bagasse, and woody biomass such as forest residue and thinned wood. Of these, herbaceous biomass such as rice straw and bagasse is generally single-year, has a short annual availability period, and has a low weight per unit volume (hereinafter referred to as volume weight) (150 or more, less than 300 kg / m ³ ). . On the other hand, woody biomass is generally perennial, so it can be said that it is available throughout the year and has a high bulk weight (300 kg / m ³ or more). In the case of herbaceous biomass, a yard for stocking raw materials that are discharged in a short period of time is necessary, whereas in woody biomass, there is no such concern, and because the bulk weight is high, the transportation cost of raw materials can be kept low. It is considered possible.

On the other hand, afforestation projects for the purpose of using pulp and building materials are being actively carried out, and it is said that the overseas afforestation area conducted by domestic companies will reach 400,000 hectares. Among them, eucalyptus planted as a pulp material accounts for the majority. Eucalyptus grows quickly and can be harvested in about 10 years, so it has already been pulped and chipped in Japan.
A tree is divided into an inner xylem and an outer bark with a formation layer where cell division is active as a boundary. In young eucalyptus, the bark has a relatively low lignin content compared to the xylem and is soft with many soluble components. In addition, the bark is divided into an outer bark of dead tissue and an inner bark of living tissue.

The outer bark mainly consists of pericytes or cork layers, which protects the wood tissue from mechanical damage and reduces fluctuations in temperature and humidity.
The inner bark consists of mentor elements, parenchyma cells and thick-walled cells. The mentor elements have the function of transporting fluids and nutrients, and the parenchyma has the function of storing nutrients such as starch and is interposed between the mentor elements of the inner bark. . Thick wall cells function as a supporting tissue, are observed in layers like the xylem rings, and are differentiated into bast fibers and scleroids by shape.

  Bark tissue is roughly divided into fine substances including fibers, cork cells, and parenchyma cells. Bark fibers are chemically similar to xylem fibers and are composed of cellulose, hemicellulose and lignin. A fine substance containing cork cells and parenchyma contains a large amount of extracted components, and the cork cell walls are rich in suberins and the fine substance fraction is rich in polyphenols. In this way, the bark contains many useful soluble components unlike the xylem, the amount reaches 20-40% of the dry mass, and the fiber fraction has the same fiber as the xylem. Has excellent properties. However, bark is not used in timber applications and can be returned to the soil as a fertilizer in the plantation along with branches and roots when pulping in the papermaking process to reduce pulp quality even if mixed slightly. They are peeled and incinerated at lumber mills or chip mills and are not effectively used as woody biomass.

As methods for converting woody biomass to bioethanol, various methods have been studied, as shown in many books (Non-patent Document 1, Non-patent Document 2, Non-patent Document 3). Among them, methods for obtaining ethanol by fermentation after mono-saccharification by acid saccharification method or enzymatic saccharification method are widely studied.
The acid saccharification method is a method of obtaining sugar by hydrolyzing woody biomass with an inorganic acid such as sulfuric acid, and a dilute acid method and a concentrated acid method have been proposed depending on the concentration (Patent Documents 1 and 2). In the dilute acid method, both the temperature and the pressure are high, and the device is corroded by the added acid. Furthermore, there is a problem that it is difficult to separate the produced saccharide and acid and there is no economically effective acid recovery method. In addition, since the concentrated acid method has a relatively low temperature and pressure, an inexpensive reactor material can be used, and the yield of glucose is high. However, there is a problem that a large amount of waste acid is generated because there is no economically effective method for separating and recovering acid from saccharides produced in the same manner as the diluted acid method.

  Enzymatic saccharification is a method in which lignocellulose is saccharified with enzymes such as cellulase and hemicellulase, and saccharification is possible under milder conditions than acid saccharification, so there is no restriction on the equipment material and the treatment of waste is simple. It is. However, since the cellulose in lignocellulose is covered with lignin and hemicellulose and the enzyme cannot easily contact the cellulose, the saccharification rate is generally low. Therefore, physical pretreatments such as fine pulverization, pressurized hot water treatment, steaming / explosion treatment, and chemical pretreatment with chemicals such as acids and alkalis have been mainly studied in order to improve the enzyme saccharification rate.

Among the physical pretreatments, pulverization is intended to promote saccharification by exfoliating part of lignin and hemicellulose covering cellulose by pulverization and increasing the frequency with which the enzyme contacts cellulose. This is a pre-processing method.
The pressurized hot water treatment is a method in which lignin and hemicellulose are softened and separated from cellulose by hot hot water. Furthermore, there is also known a method of performing saccharification with an enzyme on a processed product that has been subjected to mechanical grinding after being softened by pressurized hot water treatment (see Patent Document 3).
In addition, in the steaming / explosion treatment method in which biomass is released immediately under high temperature and high pressure treatment at atmospheric pressure or near low temperature and low pressure conditions, the cracks occur in the lignin and hemicellulose covering the cellulose. It becomes possible to contact cellulose (see Patent Document 4).

  The chemical pretreatment is a pretreatment that enables enzymatic saccharification by removing lignin and hemicellulose covering cellulose by softening or dissolving them with chemicals and exposing the cellulose to the surface. Alkaline treatment as in Patent Document 5 is a typical chemical pretreatment, but polysaccharide degrading enzymes such as cellulase and hemicellulase must be treated in a neutral to weakly acidic region, so that chemical removal is required before saccharification. Or the process of adjusting pH may be needed.

  The method of Patent Document 3 requires a large energy cost, and the method of Patent Document 4 similarly requires an energy cost and can only perform batch processing. As described above, Patent Document 5 has drawbacks such as requiring a neutralization step.

  In addition, the tissues of plants, particularly woody plants, have a composite polymer structure in which high-molecular polysaccharides such as cellulose and hemicellulose are firmly bound. Therefore, when enzymatic saccharification is performed, a method of using various hemicellulose degrading enzymes in combination with cellulase has been studied.

  As a saccharification method using a plurality of enzymes, lignocellulose is a method using an auxiliary enzyme such as cellulase and xylanase (Patent Document 6), lignocellulose (Miscanthus x giganteus) is AFEX pretreatment method (ammonia fiber explosion: ammonia fiber explosion method) ) Followed by enzymatic saccharification with cellulase and xylanase (Non-patent Document 5), after treating woody biomass with ammonia, alkali, plasticizer, etc., and then saccharifying with multiple enzymes (Patent Document 7) ) Etc.

  The method of Patent Document 6 cannot be applied to bark containing lignin. The method of Non-Patent Document 5 requires a great deal of energy. Further, Patent Document 7 has problems such as requiring vacuum equipment and a large number of chemicals.

JP 2006-75007 A JP 2006-246711 A JP 2006-136263 A JP 59-204997 A JP 2008-092910 A JP 2005-523720 A JP 2008-535664 A

Edited by the Wood Society of Japan, “Wood Biomass Utilization Technology” p19-61, published by Bunnendo, July 1997 Hideaki Yukawa et al. “Latest Biomass Energy Utilization”, Chapter II-1, CMC Publishing, August 2001 Keisuke Iizuka et al. “Latest Technology of Wood Chemicals” p6-34, CMC Publishing, published in October 2001 Funaoka et al. “New Development of Woody Organic Resources” Chapter 5-2, CMC Publishing, published in January 2005 Optimization of ammonia fiber expansion (AFEX) pretreatment and enzymatic hydrolysis of Miscanthus x giganteus to fermentable sugars. Biotechnology Progress. 23 (4): 846-50, 2007.

  In the present invention, an object of the saccharification method of bark is to provide a method that further improves the yield of saccharides and at the same time enables saccharification more easily and with low energy.

The inventors of the present invention have taken the following methods as a result of intensive studies to solve the above problems.
That is, the first of the present invention includes an alkali treatment step of immersing the bark of a woody plant in an aqueous alkali compound solution, a step of mechanically crushing the alkali-treated bark from the alkali treatment step, and the mechanical crushing. A method for saccharifying a bark of a woody plant, comprising an enzymatic saccharification treatment step of saccharifying the treated bark with an enzyme, wherein pectinase and cellulase are simultaneously used as enzymes in the enzymatic saccharification treatment step.

  A second aspect of the present invention is the woody plant saccharification method according to the first aspect of the present invention, wherein the cellulase is at least one selected from the group consisting of endoglucanase, exoglucanase, and betaglucosidase.

  In a third aspect of the present invention, the pectinase is one or more selected from the group consisting of pectin esterase, pectin lyase, pectate lyase, exopolygalacturonase, endopolygalacturonase, and rhamnogalacturonase. A method for saccharifying a woody plant according to any one of the first and second aspects of the present invention.

  According to a fourth aspect of the present invention, the alkali compound is one or more selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium oxide, sodium carbonate, sodium bicarbonate, and ammonia. A method for saccharifying bark of a woody plant according to any one of the first to third aspects of the invention.

The fifth present invention, wherein the woody plant is grayed Landis (grndis) species, globulus (globulus) species, Naitensu (nitens) species, camaldulensis (camaldulensis) species, Deguraputa (deglupta) species, Biminarisu (viminalis) The woody plant belonging to the genus Eucalyptus selected from the group consisting of a species, a europhylla species, a dunnii species and a hybrid of these species, according to any one of the first to fourth aspects of the present invention. This is a method for saccharification of woody plants.

  According to the present invention, it has become possible to provide a method for saccharification of bark that further improves the yield of saccharides and at the same time allows saccharification more easily and with low energy.

Hereinafter, embodiments of the present invention will be specifically described.
In the present invention, the bark of a woody plant is used as a biomass raw material. The species is not particularly limited, preferably eucalyptus (Eucalyptus) genus, more preferably grandis (grandis) seed, globulus (globulus) species, Naitensu (nitens) species, camaldulensis (camaldulensis) species, Deguraputa (deglupta) seed is Biminarisu (viminalis) species, Euro filler (urophylla) species, Daniai (dunnii) and their hybrids.

The bark raw material can be used as a raw material in an available state. Usually, it is preferable to treat the material as it is if it is in a state of being cut or pulverized into several cm ² in consideration of handling property at the time of conveyance. The smaller the size of the bark raw material, the easier it is to treat. However, in the method of the present invention, the bark treated in the pretreatment process can be easily refined with less energy cost by defibrating treatment, so the dried bark raw material is excessively refined It is preferable to avoid processing.

Below, the process of this invention is demonstrated in order.
The obtained raw material bark is used for the alkali treatment process immersed in aqueous alkali compound solution.
The alkali compound to be used is not particularly limited as long as it softens the bark, but sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium oxide, sodium carbonate, sodium hydrogen carbonate, ammonia and the like are preferably used. Of these, sodium hydroxide is particularly preferred.
In the alkali treatment step, any other chemicals such as sodium sulfite and sodium hydrogen sulfite can be added to the aqueous alkali compound solution as necessary.

Although the addition amount of the said alkali compound should just be able to soften a bark, it is not specifically limited, 0.1% or more is preferable with respect to dry bark mass, More preferably, it is 6 to 20%.
The treatment temperature in the alkali treatment step is not particularly limited as long as it can soften the bark, but is preferably 10 to 300 ° C, more preferably 25 to 95 ° C.
The treatment time of the alkali treatment step is not particularly limited as long as it can soften the bark, but is preferably 10 minutes to 72 hours, and more preferably 1 hour to 17 hours.

The bark that has been subjected to the alkali treatment can be subjected to the enzyme treatment step as it is, but in order to improve the enzyme reaction efficiency, it is desirable to subject it to the enzyme treatment step after undergoing a mechanical crushing treatment.
The machine used for the mechanical crushing process varies depending on the degree of the pretreatment described above, and when the lignin is sufficiently dissolved, a disintegrator that disaggregates pulp or a refiner that beats pulp can be used. Moreover, when the degree of lignin dissolution in the pretreatment is low, it is ground using a grinder or a refiner used for producing mechanical pulp from wood. As the grinder, a stone grinder that presses and grinds a lignocellulosic material against a stone roll is preferable. For example, a pocket grinder pressed by a cylinder or a chain grinder pressed by a chain can be used. As the stone, natural stone, cement stone, ceramic stone and the like can be used. A stone mill grinder can also be used.

As the refiner, various high-concentration refiner machines used for producing mechanical pulp from wood can be used. Refiner molds include a single disk refiner that is ground by a fixed disk and one rotating disk, a double disk refiner that is ground by two counter-rotating disks, and a disk that rotates on both sides of the fixed plate. A twin disc refiner can be used. Further, a conical refiner in which the rotating plate is not a flat plate but a conical shape can be used. In grinders and refiners, a grinding process is performed while preventing clogging in the grinding machine using a hot water shower or the like in order to maintain grinding efficiency.
The bark that has been subjected to alkali treatment may be subjected to a mechanical crushing treatment as it is, or may be subjected to treatment by separating the solid and liquid components into solid and liquid components by solid-liquid separation. As solid-liquid separation means, filtration under normal pressure using a filter or the like, pressure filtration, suction filtration, or centrifugal separation means can be used.

The bark treated by the above-mentioned alkali treatment step or by the mechanical crushing treatment after the alkali treatment step is directly or after washing as necessary, adjusted to a pH suitable for the enzyme treatment step as the next step, Used for processing step.
In the enzyme treatment step of the present invention, cellulase and pectinase are used as enzymes.
The cellulase can be appropriately selected from known ones and used, and is not particularly limited. For example, endoglucanase, exoglucanase, betaglucosidase, and the like can be used. It is also possible to use any combination of these.
Commercially available cellulase preparations include Trichoderma, Acremonium, Aspergillus, Phanerochaete, Trametes, Humicola, and Bacillus. ) There are cellulase preparations derived from genera and the like. Examples of such commercially available cellulase preparations include cellulosin T2 (manufactured by HIPI), mecerase (manufactured by Meiji Seika Co., Ltd.), Novozyme 188 (manufactured by Novozyme), multifect CX10L (manufactured by Genencor), and the like. Product name).

The pectinase can be appropriately selected from known ones and is not particularly limited.
For example, pectin esterase, pectin lyase, pectate lyase, exopolygalacturonase, endopolygalacturonase, and rhamnogalacturonase can be used. It is also possible to use any combination of these.
Moreover, the combination and addition ratio of the cellulase and pectinase used in the present invention are not particularly limited, but it is preferable to use a combination of cellulase and pectinase having similar action temperature and pH range.
The cellulase and pectinase used in the present invention may be commercially available preparations or those already isolated, and after being produced by organisms such as microorganisms, fungi, yeasts, bacteria, plants, etc. You may add the whole organism which produced this, without isolating by adding a liquid as it is.
Moreover, the temperature, pH, and treatment time in the enzyme treatment step are not particularly limited, and can be appropriately adjusted according to the raw materials used, the properties of the enzyme, and the like.

EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
In each Example shown below,% is mass% unless there is particular notice.

Example 1
Eucalyptus globulus bark was cut into approximately 4 cm squares and used in the following tests.
It was prepared by adding ion-exchanged water so that the total moisture content including the moisture contained in 120 g of sodium hydroxide and the water contained in the bark was 3 kg with respect to 600 g of the mass of dry bark.
This mixture was left to stand at 25 ° C. for 17 hours to give an alkali treatment.
After the alkali treatment, the bark and the alkaline solution were separated using a sieve, and this bark was further crushed using a high-concentration refiner (manufactured by Kumagai Riki Kogyo Co., Ltd.) with a clearance of 1 mm.
Next, ion-exchanged water is added so that the crushed bark has a solid content of 10%, and the pH is adjusted to 5 with concentrated sulfuric acid. Cellulase (MultifectCX10L, Genencor Kyowa), pectinase (Peclyve FR acid, manufactured by Lyven) was added to a final concentration of 2.5% and 1%, respectively, and subjected to an enzymatic saccharification treatment by shaking at a temperature of 50 ° C. and 120 rpm. After 24 hours, the total sugar concentration of the supernatant was measured. The total sugar concentration was measured by the phenol-sulfuric acid method, and the total sugar concentration was calculated from a calibration curve prepared with a glucose standard solution. The glucose concentration was measured using a biosensor (BF4, manufactured by Oji Scientific Instruments).

(Comparative Example 1)
All the treatments were carried out in the same manner as in Example 1 except that cellulase (MultifectCX10L) was used at a final concentration of 2.5% and enzymatic saccharification treatment was carried out without using pectinase, and the total sugar concentration and glucose concentration were measured in the same manner.

(Comparative Example 2)
The bark cut into 4 cm square similar to that used in Example 1 was subjected to crushing treatment with a clearance of 1 mm using a high-concentration refiner (manufactured by Kumagai Riki Kogyo) without performing alkali treatment.
Next, ion-exchanged water is added so that the crushed bark has a solid concentration of 10%, and after adjusting to pH 5 with concentrated sulfuric acid, cellulase (MultifectCX10L, Genencor Kyowa), pectinase (Peclyve FR acid, Lyven) After the enzymatic saccharification treatment was performed under the same conditions as in Example 1, the anti-total sugar concentration and glucose concentration were measured in the same manner.

  Table 1 shows the total sugar concentration and glucose concentration of Example 1 and Comparative Examples 1-2.

  From Table 1, it was shown that when the bark was enzyme-treated without alkali pretreatment, the amount of sugar produced was very low, and the enzyme reaction proceeded smoothly by the alkali treatment. Furthermore, when pectinase and cellulase were reacted at the same time, it was shown that not only total sugar production but also glucose production was improved. This is probably because the polymer complex structure in the bark was broken by pectinase, and the decomposition of cellulase into cellulose was promoted.

  Provided is a method for improving sugar production by enzymatic treatment from bark, which is a woody biomass. Sugar obtained by enzyme treatment can be supplied as a raw material for various fermentation products including bioethanol.

Claims (5)

  An alkali treatment step of immersing the bark of a woody plant in an alkaline compound aqueous solution, a step of mechanically crushing the alkali-treated bark from the alkali treatment step, and a saccharification treatment of the bark that has been mechanically crushed A method for saccharifying bark of a woody plant, comprising an enzymatic saccharification treatment step, wherein pectinase and cellulase are simultaneously used as enzymes in the enzymatic saccharification treatment step.   The method for saccharifying bark of a woody plant according to claim 1, wherein the cellulase is at least one selected from the group consisting of endoglucanase, exoglucanase, and betaglucosidase.   The pectinase is at least one selected from the group consisting of pectinesterase, pectin lyase, pectate lyase, exopolygalacturonase, endopolygalacturonase, and rhamnogalacturonase. Item 3. A method for saccharifying bark of a woody plant according to any one of Items 1 and 2.   The alkali compound is at least one selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, calcium oxide, sodium carbonate, sodium hydrogen carbonate, and ammonia. A method for saccharifying bark of a woody plant according to any one of -3. The woody plant may be a grandis, globulus, nitens, camaldulensis, degrapta, viminalis, or europhylla species. A bark of a woody plant according to any one of claims 1 to 4, which is a woody plant belonging to the genus Eucalyptus selected from the group consisting of daniii species and hybrids thereof saccharification how.